Abstract
A novel procedure for the detection of specific DNA sequences has been developed. This procedure is based on the already known method employing PCR with appropriate primers and a sequence-specific DNA probe labeled with the fluorescent agent 6-carboxylfluorescein (FAM) at the 5′ end and the fluorescence quencher BHQ-1 (black hole quencher) at the 3′ end. However, instead of the detection of the fluorescence signal with the use of real-time PCR cyclers, fluorescence/luminescence spectrometers or fluorescence polarization readers, as in all previously-reported procedures, we propose visual observation of the fluorescence under UV light directly in the reaction tube. An example for the specific detection of the Shiga toxin-producing Escherichia coli (STEC) strains, by detecting Shiga toxin genes, is demonstrated. This method appears to be specific, simple, rapid and cost effective. It may be suitable for use in research laboratories, as well as in diagnostic units of medical institutions, even those equipped only with a thermocycler and a UV transilluminator, particularly if rapid identification of a pathogen is required.
Highlights
Polymerase chain reaction (PCR) is widely used in both basic research and biotechnological or medical applications, to such an extent that it is considered a very common technique for most genetic analyses
In the procedure described in this report, we propose to detect the specific fluorescent signal by a simple observation of the reaction tube over a UV
We found that detection of such a signal is unequivocal, and the signal is specific
Summary
Polymerase chain reaction (PCR) is widely used in both basic research and biotechnological or medical applications, to such an extent that it is considered a very common technique for most genetic analyses. One of the previousy described methods for the detection of the presence of certain DNA sequences is based on the PCR reaction with appropriate primers and specific probes labeled with the fluorescent agent 6-carboxylfluorescein (FAM) at the 5′ end and the fluorescence quencher BHQ-1 (black hole quencher) at the 3′ end.
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